Sigrid A. Rajasekaran

Encapsulation of curcumin in self-assembling peptide hydrogels as injectable drug delivery vehicles.

Curcumin, a hydrophobic polyphenol, is an extract of turmeric root with antioxidant, anti-inflammatory and anti-tumorigenic properties. Its lack of water solubility and relatively low bioavailability set major limitations for its therapeutic use. In this study, a self-assembling peptide hydrogel is demonstrated to be an effective vehicle for the localized delivery of curcumin over sustained periods of time. The curcumin-hydrogel is prepared in-situ where curcumin encapsulation within the hydrogel network is accomplished concurrently with peptide self-assembly. Physical and in vitro biological studies were used to demonstrate the effectiveness of curcumin-loaded β-hairpin hydrogels as injectable agents for localized curcumin delivery. Notably, rheological characterization of the curcumin-loaded hydrogel before and after shear flow have indicated solid-like properties even at high curcumin payloads. In vitro experiments with a medulloblastoma cell line confirm that the encapsulation of the curcumin within the hydrogel does not have an adverse effect on its bioactivity. Most importantly, the rate of curcumin release and its consequent therapeutic efficacy can be conveniently modulated as a function of the concentration of the MAX8 peptide.

Estrogen Receptor-α Binds p53 Tumor Suppressor Protein Directly and Represses Its Function

Estrogen receptor-α (ERα) promotes proliferation of breast cancer cells, whereas tumor suppressor protein p53 impedes proliferation of cells with genomic damage. Whether there is a direct link between these two antagonistic pathways has remained unclear. Here we report that ERα binds directly to p53 and represses its function. The activation function-2 (AF-2) domain of ERα and the C-terminal regulatory domain of p53 are necessary for the interaction. Knocking down p53 and ERα by small interfering RNA elicits opposite effects on p53-target gene expression and cell cycle progression. Remarkably, ionizing radiation that causes genomic damage disrupts the interaction between ERα and p53. Ionizing radiation together with ERα knock down results in additive effect on transcription of endogenous p53-target gene p21 (CDKN1) in human breast cancer cells. Our findings reveal a novel mechanism for regulating p53 and suggest that suppressing p53 function is an important component in the proproliferative role of ERα.

Curcumin-induced HDAC inhibition and attenuation of medulloblastoma growth in vitro and in vivo

BackgroundMedulloblastoma is the most common brain tumor in children, and its prognosis is worse than for many other common pediatric cancers. Survivors undergoing treatment suffer from serious therapy-related side effects. Thus, it is imperative to identify safer, effective treatments for medulloblastoma. In this study we evaluated the anti-cancer potential of curcumin in medulloblastoma by testing its ability to induce apoptosis and inhibit tumor growth in vitro and in vivo using established medulloblastoma models.MethodsUsing cultured medulloblastoma cells, tumor xenografts, and the Smo/Smo transgenic medulloblastoma mouse model, the antitumor effects of curcumin were tested in vitro and in vivo.ResultsCurcumin induced apoptosis and cell cycle arrest at the G2/M phase in medulloblastoma cells. These effects were accompanied by reduced histone deacetylase (HDAC) 4 expression and activity and increased tubulin acetylation, ultimately leading to mitotic catastrophe. In in vivo medulloblastoma xenografts, curcumin reduced tumor growth and significantly increased survival in the Smo/Smo transgenic medulloblastoma mouse model.ConclusionsThe in vitro and in vivo data suggest that curcumin has the potential to be developed as a therapeutic agent for medulloblastoma.

Na,K-ATPase and epithelial tight junctions.

Tight junctions are unique organelles in polarized epithelial and endothelial cells that regulate the flow of solutes and ions across the epithelial barrier. The structure and functions of tight junctions are regulated by a wide variety of signaling and molecular mechanisms. Several recent studies in mammals, drosophila, and zebrafish reported a new role for Na,K-ATPase, a well-studied ion transporter, in the modulation of tight junction development, permeability, and polarity. In this review, we have attempted to compile these new reports and suggest a model for a conserved role of Na,K-ATPase in the regulation of tight junction structure and functions.

Therapeutic potential of curcumin in gastrointestinal diseases

Curcumin, also known as diferuloylmethane, is derived from the plant Curcuma longa and is the active ingredient of the spice turmeric. The therapeutic activities of curcumin for a wide variety of diseases such as diabetes, allergies, arthritis and other chronic and inflammatory diseases have been known for a long time. More recently, curcumins therapeutic potential for preventing and treating various cancers is being recognized. As curcumins therapeutic promise is being explored more systematically in various diseases, it has become clear that, due to its increased bioavailability in the gastrointestinal tract, curcumin may be particularly suited to be developed to treat gastrointestinal diseases. This review summarizes some of the current literature of curcumins anti-inflammatory, anti-oxidant and anti-cancer potential in inflammatory bowel diseases, hepatic fibrosis and gastrointestinal cancers.

N-glycosylation and microtubule integrity are involved in apical targeting of prostate-specific membrane antigen: implications for immunotherapy

Prostate-specific membrane antigen (PSMA) is an important biomarker expressed in prostate cancer cells with levels proportional to tumor grade. The membrane association and correlation with disease stage portend a promising role for PSMA as an antigenic target for antibody-based therapies. Successful application of such modalities necessitates a detailed knowledge of the subcellular localization and trafficking of target antigen. In this study, we show that PSMA is expressed predominantly in the apical plasma membrane in epithelial cells of the prostate gland and in well-differentiated Madin-Darby canine kidney cells. We show that PSMA is targeted directly to the apical surface and that sorting into appropriate post-Golgi vesicles is dependent upon N-glycosylation of the protein. Integrity of the microtubule cytoskeleton is also essential for delivery and retention of PSMA at the apical plasma membrane domain, as destabilization of microtubules with nocodazole or commonly used chemotherapeutic Vinca alkaloids resulted in the basolateral expression of PSMA and increased the uptake of anti-PSMA antibody from the basolateral domain. These results may have important relevance to PSMA-based immunotherapy and imaging strategies, as prostate cancer cells can maintain a well-differentiated morphology even after metastasis to distal sites. In contrast to antigens on the basolateral surface, apical antigens are separated from the circulation by tight junctions that restrict transport of molecules across the epithelium. Thus, antigens expressed on the apical plasma membrane are not exposed to intravenously administered agents. The ability to reverse the polarity of PSMA from apical to basolateral could have significant implications for the use of PSMA as a therapeutic target.

HPAF-II, a cell culture model to study pancreatic epithelial cell structure and function.

Objectives: Epithelial cells have distinct apical and basolateral plasma membrane domains separated by tight junctions. This phenotype is essential for the directional transport functions of epithelial cells. Here we characterized a well-differentiated pancreatic epithelial cell line to establish a useful model for understanding the mechanisms involved in the regulation of junctional complexes, polarity, and disease processes in the pancreas. Methods: Immunofluorescence of cell junction marker proteins and electron microscopy were used to determine the presence of tight junctions, adherens junctions, and desmosomes. The functionality of tight junctions was tested by transepithelial resistance measurements and transepithelial permeability studies of nonionic molecules. Tight junction function in polarity was determined by laser scanning confocal microscopy. Results: Immunofluorescence analysis in HPAF-II cells revealed tight junction localization of ZO-1, occludin, and claudin-4; adherens junction localization of E-cadherin and β-catenin; and desmosomal localization of desmocollin. Transmission electron microscopy showed the presence of tight junctions, adherens junctions, and des-mosomes, and freeze-fracture electron microscopy revealed the presence of distinct anastomosing tight junction strands. Transepithelial electrical resistance and permeability measurements revealed functional tight junctions. In addition, 3-dimensional images of the monolayer generated by laser scanning confocal microscopy revealed that HPAF-II cells show polarity. Immunoblotting and RT-PCR analyses revealed high expression levels of E-cadherin and Na,K-ATPase β-subunit but low levels of the transcription factor Snail in HPAF-II cells compared with MiaPaCa-2 cells. Conclusion: The HPAF-II cell line is a well-differentiated human pancreatic carcinoma cell line that should be useful as a model for studies aimed at understanding epithelial polarity, regulation of junctional complexes, and disease processes in pancreas.

α-Catenin overrides Src-dependent activation of β-catenin oncogenic signaling

Loss of α-catenin is one of the characteristics of prostate cancer. The catenins (α and β) associated with E-cadherin play a critical role in the regulation of cell-cell adhesion. Tyrosine phosphorylation of β-catenin dissociates it from E-cadherin and facilitates its entry into the nucleus, where β-catenin acts as a transcriptional activator inducing genes involved in cell proliferation. Thus, β-catenin regulates cell-cell adhesion and cell proliferation. Mechanisms controlling the balance between these functions of β-catenin invariably are altered in cancer. Although a wealth of information is available about β-catenin deregulation during oncogenesis, much less is known about how or whether α-catenin regulates β-catenin functions. In this study, we show that α-catenin acts as a switch regulating the cell-cell adhesion and proliferation functions of β-catenin. In α-catenin-null prostate cancer cells, reexpression of α-catenin increased cell-cell adhesion and decreased β-catenin transcriptional activity, cyclin D1 levels, and cell proliferation. Further, Src-mediated tyrosine phosphorylation of β-catenin is a major mechanism for decreased β-catenin interaction with E-cadherin in α-catenin-null cells. α-Catenin attenuated the effect of Src phosphorylation by increasing β-catenin association with E-cadherin. We also show that α-catenin increases the sensitivity of prostate cancer cells to a Src inhibitor in suppressing cell proliferation. This study reveals for the first time that α-catenin is a key regulator of β-catenin transcriptional activity and that the status of α-catenin expression in tumor tissues might have prognostic value for Src targeted therapy. [Mol Cancer Ther 2008;7(6):1386–97]

Prostate-specific membrane antigen associates with anaphase-promoting complex and induces chromosomal instability

Prostate-specific membrane antigen (PSMA) is a transmembrane protein highly expressed in advanced and metastatic prostate cancers. The pathologic consequence of elevated PSMA expression in not known. Here, we report that PSMA is localized to a membrane compartment in the vicinity of mitotic spindle poles and associates with the anaphase-promoting complex (APC). PSMA-expressing cells prematurely degrade cyclin B and exit mitosis due to increased APC activity and incomplete inactivation of APC by the spindle assembly checkpoint. Further, expression of PSMA in a karyotypically stable cell line induces aneuploidy. Thus, these findings provide the first evidence that PSMA has a causal role in the induction of aneuploidy and might play an etiologic role in the progression of prostate cancer. [Mol Cancer Ther 2008;7(7):2142–51]

Dysfunction of ouabain-induced cardiac contractility in mice with heart-specific ablation of Na,K-ATPase β1-subunit

Na,K-ATPase is composed of two essential alpha- and beta-subunits, both of which have multiple isoforms. Evidence indicates that the Na,K-ATPase enzymatic activity as well as its alpha(1), alpha(3) and beta(1) isoforms are reduced in the failing human heart. The catalytic alpha-subunit is the receptor for cardiac glycosides such as digitalis, used for the treatment of congestive heart failure. The role of the Na,K-ATPase beta(1)-subunit (Na,K-beta(1)) in cardiac function is not known. We used Cre/loxP technology to inactivate the Na,K-beta(1) gene exclusively in the ventricular cardiomyocytes. Animals with homozygous Na,K-beta(1) gene excision were born at the expected Mendelian ratio, grew into adulthood, and appeared to be healthy until 10 months of age. At 13-14 months, these mice had 13% higher heart/body weight ratios, and reduced contractility as revealed by echocardiography compared to their wild-type (WT) littermates. Pressure overload by transverse aortic constriction (TAC) in younger mice, resulted in compensated hypertrophy in WT mice, but decompensation in the Na,K-beta(1) KO mice. The young KO survivors of TAC exhibited decreased contractile function and mimicked the effects of the Na,K-beta(1) KO in older mice. Further, we show that intact hearts of Na,K-beta(1) KO anesthetized mice as well as isolated cardiomyocytes were insensitive to ouabain-induced positive inotropy. This insensitivity was associated with a reduction in NCX1, one of the proteins involved in regulating cardiac contractility. In conclusion, our results demonstrate that Na,K-beta(1) plays an essential role in regulating cardiac contractility and that its loss is associated with significant pathophysiology of the heart.